Investigation, management and control of a maedi outbreak in Norway in 2019-2020.

BACKGROUND: Visna-maedi is a notifiable disease in Norway, and eliminating the disease is a national goal. The import of sheep into Norway is very limited, and strict regulations apply to the movement of small ruminants between flocks and within defined geographical regions. Several outbreaks have occurred in the last 50 years, and the most recent before 2019 occurred in Trøndelag county in Central Norway in 2002. A national surveillance programme for small ruminant lentivirus infection exists since 2003. RESULTS: In 2019, the national surveillance programme detected seropositive animals for small ruminant lentivirus in a sheep flock in Trøndelag. Based on the result of polymerase chain reaction analysis and histopathological findings, the Norwegian Food Safety Authority concluded the diagnosis of maedi. Further investigations detected maedi in eight additional sheep flocks in the same county. The flocks were placed under restrictions, and the authorities also imposed restrictions on 82 contact flocks. Sequencing of partial gag genes indicated that the virus in the current outbreak was related to the small ruminant lentivirus detected in the same area between 2002 and 2005. CONCLUSIONS: The outbreak investigation shows the need for sensitive and specific diagnostic methods, and an improved and more targeted surveillance strategy. It also demonstrates the risk of disease spreading between flocks through animal movements, and highlights the importance of biosecurity and structured livestock trade. In addition to allowing livestock trade only from flocks documented free from maedi, it may be necessary to monitor sheep flocks over many years, when aiming to eliminate maedi from the Norwegian sheep population.

Differential gene expression and immune cell infiltration in maedi-visna virus-infected lung tissues.

BACKGROUND: Maedi-visna virus (MVV) is a lentivirus that infects monocyte/macrophage lineage cells in sheep, goats, and wild ruminants and causes pneumonia, mastitis, arthritis, and encephalitis. The immune response to MVV infection is complex, and a complete understanding of its infection and pathogenesis is lacking. This study investigated the in vivo transcriptomic patterns of lung tissues in sheep exposed to MVV using the RNA sequencing technology. RESULT: The results indicated that 2,739 genes were significantly differentially expressed, with 1,643 downregulated genes and 1,096 upregulated genes. Many variables that could be unique to MVV infections were discovered. Gene Ontology analysis revealed that a significant proportion of genes was enriched in terms directly related to the immune system and biological responses to viral infections. Kyoto Encyclopedia of Genes and Genomes analysis revealed that the most enriched pathways were related to virus-host cell interactions and inflammatory responses. Numerous immune-related genes, including those encoding several cytokines and interferon regulatory factors, were identified in the protein-protein interaction network of differentially expressed genes (DEGs). The expression of DEGs was evaluated using real-time polymerase chain reaction and western blot analysis. CXCL13, CXCL6, CXCL11, CCR1, CXCL8, CXCL9, CXCL10, TNFSF8, TNFRSF8, IL7R, IFN-γ, CCL2, and MMP9 were upregulated. Immunohistochemical analysis was performed to identify the types of immune cells that infiltrated MVV-infected tissues. B cells, CD4+ and CD8+ T cells, and macrophages were the most prevalent immune cells correlated with MVV infection in the lungs. CONCLUSION: Overall, the findings of this study provide a comprehensive understanding of the in vivo host response to MVV infection and offer new perspectives on the gene regulatory networks that underlie pathogenesis in natural hosts.

Next-generation sequencing for the genetic characterization of Maedi/Visna virus isolated from the northwest of China.

BACKGROUND: Maedi/Visna virus (MVV) is a contagious viral pathogen that causes considerable economic losses to the sheep industry worldwide. OBJECTIVES: In China, MVV has been detected in several regions, but its molecular characteristics and genetic variations were not thoroughly investigated. METHODS: Therefore, in this study, we conducted next-generation sequencing on an MVV strain obtained from northwest China to reveal its genetic evolution via phylogenetic analysis. RESULTS: A MVV strain obtained from Inner Mongolia (NM) of China was identified. Sequence analysis indicated that its whole-genome length is 9193 bp. Homology comparison of nucleotides between the NM strain and reference strains showed that the sequence homology of gag and env were 77.1%-86.8% and 67.7%-75.5%, respectively. Phylogenetic analysis revealed that the NM strain was closely related to the reference strains isolated from America, which belong to the A2 type. Notably, there were 5 amino acid insertions in variable region 4 and a highly variable motif at the C-terminal of the surface glycoprotein (SU5). CONCLUSIONS: The present study is the first to show the whole-genome sequence of an MVV obtained from China. The detailed analyses provide essential information for understanding the genetic characteristics of MVV, and the results enrich the MVV library.

Species-Specific Humoral Immune Responses in Sheep and Goats upon Small Ruminant Lentivirus Infections Inversely Correlate with Protection against Virus Replication and Pathological Lesions.

Maedi-Visna-like genotype A strains and Caprine arthritis encephaltis-like genotype B strains are small ruminant lentiviruses (SRLV) which, for incompletely understood reasons, appear to be more virulent in sheep and goats, respectively. A 9-month in vivo infection experiment using Belgian genotype A and B SRLV strains showed that almost all homologous (genotype A in sheep; genotype B in goats) and heterologous (genotype A in goats; genotype B in sheep) intratracheal inoculations resulted in productive infection. No differences in viremia and time to seroconversion were observed between homologous and heterologous infections. Higher viral loads and more severe lesions in the mammary gland and lung were however detected at 9 months post homologous compared to heterologous infection which coincided with strongly increased IFN-γ mRNA expression levels upon homologous infection. Pepscan analysis revealed a strong antibody response against immune-dominant regions of the capsid and surface proteins upon homologous infection, which was absent after heterologous infection. These results inversely correlated with protection against virus replication in target organs and observed histopathological lesions, and thus require an in-depth evaluation of a potential role of antibody dependent enhancement in SRLV infection. Finally, no horizontal intra- and cross-species SRLV transmission to contact animals was detected.

Epidemiology and control of maedi-visna virus: Curing the flock.

Maedi-visna (MV) is a complex lentiviral disease syndrome characterised by long immunological and clinical latencies and chronic progressive inflammatory pathology. Incurable at the individual level, it is widespread in most sheep-keeping countries, and is a cause of lost production and poor animal welfare. Culling seropositive animals is the main means of control, but it might be possible to manage virus transmission effectively if its epidemiology was better quantified. We derive a mathematical epidemiological model of the temporal distributions of seroconversion probabilities and estimate susceptibility, transmission rate and latencies in three serological datasets. We demonstrate the existence of epidemiological latency, which has not explicitly been recognised in the SRLV literaure. This time delay between infection and infectiousness apparently exceeds the delay between infection and seroconversion. Poor body condition was associated with more rapid seroconversion, but not with a higher probability of infection. We estimate transmission rates amongst housed sheep to be at about 1,000 times faster than when sheep were at grass, when transmission was negligible. Maternal transmission has only a small role in transmission, because lambs from infected ewes have a low probability of being infected directly by them, and only a small proportion of lambs need be retained to maintain flock size. Our results show that MV is overwhelmingly a disease of housing, where sheep are kept in close proximity. Prevalence of MV is likely to double each year from an initial low incidence in housed flocks penned in typically-sized groups of sheep (c. 50) for even a few days per year. Ewes kept entirely at grass are unlikely to experience transmission frequently enough for MV to persist, and pre-existing infection should die out as older ewes are replaced, thereby essentially curing the flock.

Characterization of minimal lesions related to the presence of visna/maedi virus in the mammary gland and milk of dairy sheep.

BACKGROUND: In order to characterize the complete range of lesions, especially minimal, affecting mammary gland and viral antigen distribution and target cells using immunohistochemistry in naturally Visna/maedi (VM) 84 infected sheep were studied, forty-four from flocks with clinical cases (A) and 35 randomly sampled from two abattoirs (B) together with five negative controls (C). An immunocytochemistry technique was developed and further milk samples (n = 39) were used to study viral excretion, carrier cells and the role of milk and colostrum in the transmission of the disease. RESULTS: All sheep from group C and three sheep from group B were negative to VM in tissue sections by histopathology, immunohistochemistry and PCR, and also in serum using ELISA. Several degrees of CD3 + lymphocytic interstitial mastitis were observed in groups A and B: minimal (+) n = 26 sheep; moderate (++), n = 32 and severe (+++), n = 12. No differences in lesion distribution were observed between groups A and B. Viral presence was confirmed by immunohistochemistry using two different antibodies and/or PCR in every tissue with lesions while serology was negative in six sheep with lesions. Two milk samples taken from milk tanks from two flocks from group A and fourteen milk samples from 29 infected sheep from group B were positive to VM (most of them from animals with moderate and severe lesions). Positivity was only found in macrophages, even in focal and minimal lesions, while no positivity was observed in epithelial or any other cells in either tissue and milk samples. CONCLUSIONS: This new observation of the minimal lesions described in this work increased the prevalence of VM lesions in mammary gland up to 90.9% and VM should be considered as a differential diagnosis when minimal interstitial lesions are detected. A high prevalence of VM was observed in intensive milk-producing sheep, ELISA serology did not detect as positivity all infected animals, while histology, IHC or PCR showed higher sensitivity. The cytological technique developed was very useful in milk-cell studies using hematoxylin and eosin and immunocytochemistry. Viral detection in milk samples (16/39) confirms a potential but limited role of milk/colostrum in viral transmission.

First survey on association of TMEM154 and CCR5 variants with serological maedi-visna status of sheep in German flocks.

Maedi-visna, a disease caused by small ruminant lentiviruses (SRLVs), is present in sheep from many countries, also including Germany. An amino acid substitution (E/K) at position 35 of the transmembrane protein 154 (TMEM154) as well as a deletion in the chemokine (C-C motif) receptor type 5 gene (CCR5) were reported to be associated with the serological MV status and/or the SRLV provirus concentration in North American sheep populations. The aim of this study was to test if those two gene variants might be useful markers for MV susceptibility in Germany. For this purpose, more than 500 sheep from 17 serologically MV positive German sheep flocks with different breed backgrounds were genotyped applying PCR-based methods. Both, crosstab and non-parametric analyses showed significant associations of the amino acid substitution at position 35 of TMEM154 with the serological MV status (cut-off-based classification) and the median MV ELISA S/P value in all samples and in two of the four analyzed breed subsets. The deletion in the CCR5 promoter did not show a consistent association with serological MV status or median ELISA S/P value. It can be concluded that the amino acid substitution at position 35 of TMEM154 is a promising marker for breeding towards a lower number of serologically MV positive sheep in German flocks, at least in flocks of the Texel breed, while this remains questionable for the deletion in the CCR5 promoter. The findings of this study still need to be verified in additional sheep breeds.

Maedi-visna virus persistence: Antigenic variation and latency.

Maedi-visna virus (MVV), a lentivirus of sheep, shares with other lentiviruses the ability to establish a lifelong infection. In this study five sheep were infected intravenously with MVV and housed together with a number of uninfected sheep for natural transmission. All virus isolates from ten sheep that had been infected naturally had multiple mutations in the principal neutralization domain in Env and were antigenic variants, while three of four isolates from the carrier sheep had identical sequences to the infecting strain and were not antigenic variants. There was evidence of positive selection in the gene, particularly in amino acids comprising the neutralization epitope and some adjacent glycosylation sites. Together these results suggest that virus persistence is acquired by a reservoir of latent viruses, and that there is selection for antigenic variants of virus that is transmitted naturally.

Prevalence of Maedi-visna in Saskatchewan sheep.

A study was conducted to estimate flock and individual seroprevalence of Maedi-visna in Saskatchewan and evaluate risk factors for seropositive flocks. Thirty-five percent (24/68) of flocks and 4.6% (93/2010) of individual samples were positive. Within-flock prevalence ranged from 3.3% to 96.7%. Significant flock-level predictors of flock prevalence included large flock size, purchasing > 50 sheep and respiratory problems in the previous 5 years.

Prévalence de maedi-visna chez des moutons de la Saskatchewan. Une étude a été réalisée pour estimer la séroprévalence individuelle et dans le troupeau de maedi-visna en Saskatchewan et évaluer les facteurs de risque des troupeaux séropositifs. Trente-cinq pour cent (24/68) des échantillons des troupeaux et 4,6 % (93/2010) des échantillons individuels étaient positifs. La prévalence dans le troupeau variait de 3,3 % à 96,7 %. Les prédicteurs importants au niveau du troupeau incluaient une taille importante du troupeau, l'achat de > 50 moutons et des problèmes respiratoires au cours des cinq années antérieures.(Traduit par Isabelle Vallières).

A census to determine the prevalence and risk factors for caprine arthritis-encephalitis virus and visna/maedi virus in the Swiss goat population.

In Switzerland, viruses belonging to two different phylogenetic groups of small ruminant lentiviruses (SRLV) are currently circulating: the caprine arthritis-encephalitis virus (CAEV) and visna/maedi virus (VMV). In the past two decades, a mandatory national control program has led to a very low prevalence of seropositivity, while completely eliminating CAE as a clinical manifestation. However, in order to reduce the high costs and effort associated with this program, adjustments based on the most recent epidemiological knowledge are needed. The purpose of this study was to estimate the seroprevalence of CAEV and VMV using the newest diagnostic tools available, and to identify potential risk factors for infection with these viruses in Switzerland. For the prevalence estimation, a census was carried out including 10,696 farms with a total of 85,454 goats. Blood samples were analysed using a 3-step serological testing algorithm consisting of Chekit ELISA, Western Blot and SU5 ELISA. A risk factor analysis was conducted using logistic regression models built with data obtained from a mail questionnaire, and serological results from the census. The apparent herd-level prevalences were 0.38%, 2.77%, and 3.04% for CAEV, VMV and SRLV, respectively. Animal-level prevalences were 0.06% for CAEV, 0.55% for VMV, and 0.61% for SRLV. No statistically significant risk factors associated with CAEV or VMV infection were identified. However, the proportional high number of CAEV seropositive dwarf goats, in relation to their population size, could indicate that these hobby breeds may slip through some of the official controls. For an infection with SRLV, a medium herd size (7-40 goats) was found to be protective, compared with smaller (OR=1.90, p=0.034) and larger herds (OR=1.95, p=0.038). In conclusion, considering that all CAEV positive animals were culled, these results imply that CAEV is no longer actively spreading and has successfully been controlled in Switzerland. However, given the uncertain pathogenic potential of VMV in goats, future surveillance should also be taking into account the not insignificant number of VMV circulating in the Swiss goat population.

The occurrence of maedi-visna virus in Lebanon.

Maedi-visna (MV) is a chronic viral disease prevalent in adult sheep that is caused by a virus belonging to the small ruminant lentivirus group (SRLV). This disease is considered to affect the international trade of sheep and is classified in the World Organisation for Animal Health (OIE) list of notifiable animal diseases. Although maedi-visna virus (MVV) has been detected in many countries, no study on its occurrence has been carried out in Lebanon. For this purpose, a serological survey of infection with MVV was conducted in seven of the eight Lebanese governorates using a competitive enzyme-linked immunosorbent assay (ELISA). A total of 184 individual blood samples from sheep of the local breed 'Awassi', originating from 16 farms distributed throughout the seven Lebanese governorates, were collected and analysed. Among the 184 tested sheep, 131 sheep from the16 farms visited were MVV positive. This presents a prevalence of 71% MVV-positive animals and 100% MVV-positive farms. The results indicate the need for further systematic investigations into the between-herd and within-herd prevalence of MV in Lebanon.

Le maedi-visna (MV) est une maladie virale chronique causée par un virus appartenant au groupe des lentivirus des petits ruminants (SRLV) et affectant les moutons adultes. La maladie a une incidence sur les échanges internationaux d'ovins et figure sur la liste des maladies à déclaration obligatoire de l'Organisation mondiale de la santé animale (OIE). La présence du virus maedi-visna est attestée dans de nombreux pays mais jusqu'à présent aucune étude ne lui avait été consacrée au Liban. Pour y remédier, une enquête sérologique recourant à une épreuve immuno-enzymatique (ELISA) par compétition a été conduite dans sept des huit gouvernorats du Liban afin de déterminer la prévalence de l'infection par le virus maedi-visna. Au total, 184 échantillons sanguins prélevés sur des moutons de race locale (Awassi) originaires de 16 élevages répartis dans les sept gouvernorats ont été analysés. Des résultats positifs ont été obtenus sur 131 des 184 prélèvements ; tous les élevages étaient représentés. La prévalence est donc de 71 % à l'échelle des individus et de 100 % à l'échelle des élevages. Il ressort de ces résultats que la prévalence à l'intérieur des élevages ainsi que celle entre élevages devraient faire l'objet d'enquêtes systématiques plus poussées au Liban.

El maedi-visna (MV) es una enfermedad viral crónica prevalente en ovejas adultas, cuyo agente etiológico es un virus del grupo de los lentivirus de los pequeños rumiantes. Figura en la lista de enfermedades de declaración obligatoria de la Organización Mundial de Sanidad Animal (OIE) porque se considera que afecta al comercio internacional de ovejas. Aunque el virus maedi-visna (VMV) ha sido detectado en muchos países, nunca antes se había estudiado su presencia en el Líbano. Para ello se llevó a cabo un estudio serológico de la infección por el virus en siete de los ocho distritos administrativos del país mediante un ensayo inmunoenzimático (ELISA) de competición. Se extrajeron y analizaron un total de 184 muestras sanguíneas de ovejas de la raza autóctona «awassi¼ procedentes de 16 explotaciones repartidas en los siete distritos libaneses. De esas 184 muestras, dieron resultado positivo para el VMV 131, correspondientes a ovejas de las 16 explotaciones visitadas. Ello supone una prevalencia del 71% de animales positivos al virus y del 100% de explotaciones positivas. Los resultados ponen de manifiesto la necesidad de investigar más sistemáticamente la prevalencia del maedi-visna entre los rebaños y dentro de los rebaños del Líbano.

Modulation of the long terminal repeat promoter activity of small ruminant lentiviruses by steroids.

Production and excretion of small ruminant lentiviruses (SRLVs) varies with the stage of the host reproductive cycle, suggesting hormonal involvement in this variation. Stress may also affect viral expression. To determine if hormones affect SRLV transcriptional activity, the expression of green fluorescent protein (GFP) driven by the promoters in the U3-cap region of the long terminal repeats (LTRs) of different strains of SRLV was assessed in cell culture. High concentrations of steroids (progesterone, cortisol and dehydroepiandrosterone) inhibited expression of GFP driven by SRLV promoters. This effect decreased in a dose-dependent manner with decreasing concentrations of steroids. In some strains, physiological concentrations of cortisol or dehydroepiandrosterone (DHEA) induced the expression of GFP above the baseline. There was strain variation in sensitivity to hormones, but this differed for different hormones. The presence of deletions and a 43 base repeat in the U3 region upstream of the TATA box of the LTR made strain EV1 less sensitive to DHEA. However, no clear tendencies or patterns were observed when comparing strains of different genotypes and/or subtypes, or those triggering different forms of disease.

Mutations in Ovis aries TMEM154 are associated with lower small ruminant lentivirus proviral concentration in one sheep flock.

Small ruminant lentivirus (SRLV), also called ovine progressive pneumonia virus or maedi-visna, is present in 24% of US sheep. Like human immunodeficiency virus, SRLV is a macrophage-tropic lentivirus that causes lifelong infection. The production impacts from SRLV are due to a range of disease symptoms, including pneumonia, arthritis, mastitis, body condition wasting and encephalitis. There is no cure and no effective vaccine for preventing SRLV infection. However, breed differences in prevalence and proviral concentration indicate a genetic basis for susceptibility to SRLV. Animals with high blood proviral concentration show increased tissue lesion severity, so proviral concentration represents a live animal test for control post-infection in terms of proviral replication and disease severity. Recently, it was found that sheep with two copies of TMEM154 haplotype 1 (encoding lysine at position 35) had lower odds of SRLV infection. In this study, we examined the relationship between SRLV control post-infection and variants in two genes, TMEM154 and CCR5, in four flocks containing 1403 SRLV-positive sheep. We found two copies of TMEM154 haplotype 1 were associated with lower SRLV proviral concentration in one flock (P < 0.02). This identified the same favorable diplotype for SRLV control post-infection as for odds of infection. However, frequencies of haplotypes 2 and 3 were too low in the other three flocks to test. The CCR5 promoter deletion did not have consistent association with SRLV proviral concentration. Future work in flocks with more balanced allele frequencies is needed to confirm or refute TMEM154 association with control of SRLV post-infection.

Successful Visna/maedi control in a highly infected ovine dairy flock using serologic segregation and management strategies.

A control system for Visna/maedi virus (VMV) infection based on serologic segregation and management strategies was applied in an infected Spanish dairy Manchega breed sheep flock (n=670) that was affected by a severe respiratory process associated to VMV. The control started in 2004 and consisted on the serological study of animals, segregation in two different flocks (seropositive and seronegative), separate management of flocks, selection of young female lambs for replacement only from seronegative ewes offspring, immediate removal of seropositive animals detected in the seronegative flock and a management tending toward the reduction and final culling of the seropositive flock. The serological control was repeated yearly or twice a year, approximately. Initial VMV seroprevalence of the undivided flock was 66.4% (January 2004) that descended to 47.3%, 12.8%, 2.2% and 0.2% between July 2004 and May 2006. Residual seroprevalence fluctuated slightly thereafter with a peak of 2.2% in April 2008. After segregation, number of animals in the seronegative flock was 378 that descended to 323 in October 2005. Since then, this number has increased steadily reaching 650 sheep in December 2011. The seropositive flock was progressively reduced by culling until its total disappearance in June 2010. This work presents the detailed results obtained in the control strategy against VMV in a single dairy sheep flock by implementing a segregation system based on serologic testing. The system is highly successful, as it reduces to residual levels VMV infection in about two years without the need of culling a high number of animals, as required by other methods. Moreover, the original size flock was been recovered within 8 years and has led to a subjective improvement of animal health and welfare in the flock. The residual seroprevalence could be eliminated at this stage by applying more sensitive molecular or other serological techniques to reach eradication.

Impact of maedi-visna in intensively managed dairy sheep.

Maedi-visna (MV) is a slow lentiviral disease of sheep that has a significant economic impact in many sheep-producing regions although there remains a paucity of data relating to actual production losses resulting from this disease. The objective of this study was to evaluate direct losses, through death or culling, from two dairy sheep flocks with high seroprevalences of infection over a 2 year period. Maedi-visna was found, either alone or in combination with other diseases, to be the most common disease diagnosed in these sheep, and the major cause of direct animal losses in the two flocks. Moderate to severe lesions associated with MV were found in 52% and 80% of the sheep, respectively, affecting the lungs, brain and/or mammary glands. Despite the similarity of the two flocks under study in terms of breed, number of animals, geographical proximity, and inter-change of rams, a striking difference was observed regarding the clinical presentation of the disease: in one flock the respiratory form was dominant while in the other 70% of animals died or were culled because of neurological signs.

Expression analysis of 13 ovine immune response candidate genes in Visna/Maedi disease progression.

Visna/Maedi virus (VMV) is a lentivirus that infects cells of the monocyte/macrophage lineage in sheep. Infection with VMV may lead to Visna/Maedi (VM) disease, which causes a multisystemic inflammatory disorder causing pneumonia, encephalitis, mastitis and arthritis. The role of ovine immune response genes in the development of VM disease is not fully understood. In this work, sheep of the Rasa Aragonesa breed were divided into two groups depending on the presence/absence of VM-characteristic clinical lesions in the aforementioned organs and the relative levels of candidate gene expression, including cytokines and innate immunity loci were measured by qPCR in the lung and udder. Sheep with lung lesions showed differential expression in five target genes: CCR5, TLR7, and TLR8 were up regulated and IL2 and TNFα down regulated. TNFα up regulation was detected in the udder.

Small ruminant lentiviruses in Jordan: evaluation of sheep and goat serological response using recombinant and peptide antigens.

Small ruminant lentiviruses infect sheep and goats worldwide, causing chronic progressive diseases and relevant economic losses. Disease eradication and prevention is mostly based on serological testing. The goal of this research was to investigate the presence of the small ruminant lentiviruses (SRLVs) in Jordan and to characterize the serological response in sheep and goat populations. A panel of sera were collected from flocks located in Northern Jordan and Jordan Valley. The samples were tested using three ELISA assays: a commercially available ELISA based on p25 recombinant protein and transmembrane peptide derived from British maedi-visna virus (MVV) EV1 strain, an ELISA based on P16-P25 recombinant protein derived from two Italian strains representative of MVV- and caprine arthritis encephalitis virus (CAEV)-like SRLVs, and an ELISA based on SU5 peptide from the same two Italian isolates. The results indicate that both MVV- and CAEV-like strains are present in Jordan and that the majority of the viruses circulating among sheep and goat populations belong to the MVV-like genotype.

First molecular characterization of visna/maedi viruses from naturally infected sheep in Turkey.

Recent worldwide serological and genetic studies of small ruminant lentiviruses (SRLV) have led to the description of new genotypes and the development of new diagnostic tests. This study investigated the detection and molecular characterization of visna/maedi virus (VMV) infection in serum and blood samples from pure and mixed sheep breeds acquired from different regions in Turkey using ELISA and PCR techniques. The prevalence of VMV was 67.8 % by ELISA and/or LTR-PCR with both assays showing a medium level of agreement (kappa: 0.26; ± 0.038 CI). Positivity of VMV in sheep increased according to the age of the animal, although PCR positivity was higher than ELISA in young individuals. Phylogenetic analysis of 33 LTR sequences identified two distinct clades that were closely related to American and Greek LTR sequences. Phylogenetic analysis of 10 partial gag gene sequences identified A2, A3, A5, A9, A11 subtypes of genotype A SRLVs. In vitro culture of all isolates in fetal sheep lung cells (FSLC) showed a slow/low phenotype causing less or no lytic infection compared with infection with the WLC-1 American strain characterized by a rapid/highly lytic phenotype. Phylogenetic analysis revealed that Turkish VMV sequences preceded the establishment of American or Greek strains that were associated with the migration of sheep from the Middle East to Western Europe several centuries ago. This is the first study that describes Turkish VMV sequences with the molecular characterization of LTR and gag genes, and it strongly suggests that SRLV-genotype A originated in Turkey.

Reduced lentivirus susceptibility in sheep with TMEM154 mutations.

Visna/Maedi, or ovine progressive pneumonia (OPP) as it is known in the United States, is an incurable slow-acting disease of sheep caused by persistent lentivirus infection. This disease affects multiple tissues, including those of the respiratory and central nervous systems. Our aim was to identify ovine genetic risk factors for lentivirus infection. Sixty-nine matched pairs of infected cases and uninfected controls were identified among 736 naturally exposed sheep older than five years of age. These pairs were used in a genome-wide association study with 50,614 markers. A single SNP was identified in the ovine transmembrane protein (TMEM154) that exceeded genome-wide significance (unadjusted p-value 3×10(-9)). Sanger sequencing of the ovine TMEM154 coding region identified six missense and two frameshift deletion mutations in the predicted signal peptide and extracellular domain. Two TMEM154 haplotypes encoding glutamate (E) at position 35 were associated with infection while a third haplotype with lysine (K) at position 35 was not. Haplotypes encoding full-length E35 isoforms were analyzed together as genetic risk factors in a multi-breed, matched case-control design, with 61 pairs of 4-year-old ewes. The odds of infection for ewes with one copy of a full-length TMEM154 E35 allele were 28 times greater than the odds for those without (p-value<0.0001, 95% CI 5-1,100). In a combined analysis of nine cohorts with 2,705 sheep from Nebraska, Idaho, and Iowa, the relative risk of infection was 2.85 times greater for sheep with a full-length TMEM154 E35 allele (p-value<0.0001, 95% CI 2.36-3.43). Although rare, some sheep were homozygous for TMEM154 deletion mutations and remained uninfected despite a lifetime of significant exposure. Together, these findings indicate that TMEM154 may play a central role in ovine lentivirus infection and removing sheep with the most susceptible genotypes may help eradicate OPP and protect flocks from reinfection.

Small ruminant lentiviruses: immunopathogenesis of visna-maedi and caprine arthritis and encephalitis virus.

The small ruminant lentiviruses include the prototype for the genus, visna-maedi virus (VMV) as well as caprine arthritis encephalitis virus (CAEV). Infection of sheep or goats with these viruses causes slow, progressive, inflammatory pathology in many tissues, but the most common clinical signs result from pathology in the lung, mammary gland, central nervous system and joints. This review examines replication, immunity to and pathogenesis of these viruses and highlights major differences from and similarities to some of the other lentiviruses.